Quantitative Determination of DNA–Ligand Binding Using Fluorescence Spectroscopy

DAPI, 4′,6-diamidino-2-phenylindole, binds to double-stranded DNA forming a complex that fluoresces up to twenty times more than DAPI alone. It is widely used in biochemical and cytochemical studies for DNA detection. The experiment described here measures the DAPI binding constant, K ƒ, for calf-thymus DNA using fluorescence measurements and a modified Scatchard analysis. This experiment also allows the student to estimate the occupancy of the minor groove by calculating the number of nucleotides per bound ligand. Modern pharmaceutical studies involve the generation of fluorescence titration curves by the addition of aliquots of DNA to a fixed quantity of the therapeutic agent or ligand. A Scatchard plot of the binding data is then calculated by plotting the ratio of bound/free ligand versus the bound fraction and calculating the slope. This experiment utilizes the same one-cuvette-type procedure, but employs a modified Scatchard analysis that has the student calculate a fraction, ƒ, that represents the fraction of bound ligand varying from zero to one. The modified Scatchard plot involves a reciprocal plot of DNA/ƒ versus 1/(1 − ƒ ), where the slope yields the binding constant K ƒ and the intercept gives a value for the number of nucleotides per ligand binding site.